Leishmania donovani promastigotes evade the activation of mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase-1/2 during infection of naive macrophages

The protozoan parasite Leishmania fails to activate naive macrophages for proinflammatory cytokines production, and selectively impairs signal transduction pathways in infected macrophages. Because mitogen-activated protein kinases (MAPK)- and NF-kappaB-dependent signaling pathways regulate proinflammatory cytokines release, we investigated their activation in mouse bone marrow-derived macrophages (BMM) exposed to Leishmania donovani promastigotes. In naive BMM, the parasite failed to induce the phosphorylation of p38 MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)1/2, as well as the degradation of IkappaB-alpha. The use of L. donovani mutants defective in the biosynthesis of lipophosphoglycan revealed that evasion of ERK1/2 activation requires surface expression of the repeating unit moiety of this virulence determinant. In IFN-gamma-primed BMM, L. donovani promastigotes strongly induced the phosphorylation of p38 MAPK and ERK1/2, and the use of selective inhibitors for ERK (PD98059) and p38 MAPK (SB203580) revealed that both kinases are required for L. donovani-induced TNF-alpha but not NO(2)(-) release. Collectively, these data suggest that both p38 MAPK and ERK1/2 pathways participate in some Leishmania-induced responses in IFN-gamma-primed BMM. The ability of L. donovani promastigotes to avoid MAPK and NF-kappaB activation in naive macrophages may be part of the strategy evolved by this parasite to evade innate immune responses.     

Nerve growth factor regulates TNF-alpha production in mouse macrophages via MAP kinase activation

In this study, we examined the expression of nerve growth factor (NGF) and its receptors in mouse macrophages and the mechanisms involved in the effect of NGF on tumor necrosis factor (TNF)-alpha production. Macrophages expressed NGF and the NGF receptors TrkA and p75. Treatment of J744 cells or peritoneal macrophages with NGF induced a large increase in the production of TNF-alpha. In addition, NGF induced the secretion of nitric oxide in interferon-gamma-treated J774 cells or lipopolysaccharide-treated peritoneal macrophages. The induction of TNF-alpha production by NGF was blocked by K252a, an inhibitor of the TrkA receptor. NGF induced phosphorylation and activation of extracellular signal-regulated kinase, Erk1/Erk2 and c-Jun amino-terminal kinase, whereas it did not induce phosphorylation of p38 mitogen-activated protein kinase. Inhibition of the MAP kinase-Erk kinase pathway with PD 098059 decreased the secretion of TNF-alpha by NGF. Our results suggest that NGF has an important role in the activation of macrophages during inflammatory responses via activation of mitogen-activated protein kinases.     

Insulin-like growth factor-I inhibits cell growth in the a549 non-small lung cancer cell line

Insulin-like growth factors (IGFs) are potent mitogenic and antiapoptotic factors for many cell types, including some normal and neoplastic lung cells in vitro. However, in this study we show that IGF-I, at concentrations of 10 ng/ml or greater, significantly inhibits DNA synthesis and cell proliferation in a human lung adenocarcinoma cell line, A549. Inhibition of DNA synthesis was completely reversed by an IGF-I receptor-neutralizing antibody, alphaIR-3, indicating that IGF-I receptor activation is involved in its inhibitory effect. Attenuation of the p44/42 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways downstream of the IGF-I receptor using the inhibitors PD98059 and LY294002, respectively, partially reversed IGF-I-induced inhibition. Acute (2-60 min) and chronic (24 h) exposure of A549 cells to 100 ng/ml IGF-I resulted in sustained phosphorylation of Akt/protein kinase B downstream of PI 3'-kinase, whereas p44/42 MAPK phosphorylation was decreased in response to chronic exposure to IGF-I. An IGF-I dose-dependent increase in the cyclin-dependent kinase inhibitor p21(Cip1/WAF1) was also observed over 24 h of treatment. Collectively, these data suggest that IGF-I is growth inhibitory to A549 cells, possibly via sustained activation of the PI 3'-kinase signaling pathway, and induction of p21(Cip1/WAF1).     

Differential activation of MAPK signaling pathways by TGF-beta1 forms the molecular mechanism behind its dose-dependent bidirectional effects on hematopoiesis

We have earlier reported that transforming growth factor-beta1 (TGF-beta1), a well-known inhibitor of hematopoiesis, stimulated colony formation from adult human bone marrow mononuclear cells (BM MNC) when used at low concentrations. We examined the possible molecular mechanism behind this bidirectional effect using CD34+ cells isolated from human BM for clonal assays and the KG1a cell line as a model system for analysis of proteins for signaling pathways by immunoblotting. We found that TGF-beta1 at low doses (picogram levels) stimulated the colony formation from CD34+ cells, indicating that these progenitors form the direct target of stimulatory action of TGF-beta1. CD34+ cells were found to be more sensitive to the TGF-beta1 concentration than the total MNC. We used the KG1a cell line as a model system for identification of mitogen-activated protein kinase (MAPK) and AKT signaling pathways involved in the process. Low doses strongly induced p44/42 MAPK phosphorylation, whereas high doses induced p38 activation. Use of specific p44/42 MAPK inhibitor PD 98059 in the colony assay abrogated the stimulatory effect of low TGF-beta1. On the other hand, use of p38 MAPK inhibitor SB 203580 along with low TGF-beta1 concentrations had a synergistic effect on stimulation of colony formation. Treatment of BM MNC with Anisomycin, which activates stress kinases, resulted in a dose-dependent inhibition of colony formation. This inhibition could not be rescued by stimulatory doses of TGF-beta1. Phosphorylation of AKT was found to occur in a dose-dependent way but declined slightly at the highest concentration used (10 ng/ml). Inhibition of the AKT pathway by LY 294002 strongly suppressed colony formation. These data indicate clearly that sustained activation of p44/42 MAPK perhaps forms the stimulatory signal induced by low TGF-beta1, whereas activation of p38 forms the inhibitory pathway.     

Inhibition of monocytic interleukin-12 production by Candida albicans via selective activation of ERK mitogen-activated protein kinase

Our previous data demonstrated that live Candida albicans inhibits interleukin-12 (IL-12) production by human monocytes. Here we explored whether C. albicans inhibits IL-12 via a released factor and whether the inhibition is mediated via mitogen-activated protein kinase (MAPK) regulation. Supernatant fluids were obtained from cultured C. albicans (SC5314) as well as cultured Saccharomyces cerevisiae after 20 h of incubation. At 2 h postincubation of monocytes with heat-killed C. albicans (HKCA) (2:1) to stimulate IL-12, concentrated fungal supernatant fluids were added and incubated for an additional 20 h. The present data show that, unlike supernatant fluids obtained from S. cerevisiae, the C. albicans supernatant fluids significantly suppressed IL-12 production induced by HKCA. This suggested that the inhibition is Candida specific. A preliminary biochemical analysis revealed that this secretory IL-12 inhibitory factor is glycoprotein in nature. The inhibitory activity had no effect on the phagocytosis of yeasts. Supernatant fluids from C. albicans markedly induced the phosphorylation of ERK44/42 MAPK, but not p38 and SAPK, 1 min after they were added to monocytes. To test if the induction of ERK44/42 MAPK was central to the IL-12 inhibition, we used gamma interferon (IFN-gamma) (1 ng/ml) plus lipopolysaccharide (LPS) (100 ng/ml) to stimulate IL-12 production by monocytes. The inhibition of ERK MAPK by the specific inhibitor PD 98059 significantly reduced phospho-ERK44/42 MAPK levels induced by C. albicans supernatant fluids in the IFN-gamma-plus-LPS-driven monocytes. Concomitantly, PD 98059 reversed the IL-12 inhibitory activity of the C. albicans supernatant (P < 0.01). These data indicate that C. albicans can inhibit IL-12 production by secreting an ERK44/42 MAPK-stimulating factor and thus can attenuate effective immune responses.     

beta-hexosaminidase-induced activation of p44/42 mitogen-activated protein kinase is dependent on p21Ras and protein kinase C and mediates bovine airway smooth-muscle proliferation.

Late-phase and sustained activation of p44/42(MAPK) has been reported to be a critical factor in cell mitogenesis. We therefore hypothesized that p44/42(MAPK) is involved in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth-muscle cells (ASMC). Treatment of adherent ASMC with beta-hexosaminidase A (Hex A, 50 nM), an endogenous mannosyl-rich glycoprotein, resulted in a late-onset (30-min) activation of p44/42(MAPK) that lasted for 4 h. Activation of p44/42(MAPK) induced by Hex A was inhibited by an 18-mer phosphorothioate-derivatized antisense oligonucleotide (1-5 microM) directed to human p44(MAPK); the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059 (5 microM); the p42(MAPK) inhibitor Tyrphostin AG-126 (0.2 microM); the farnesyl transferase inhibitors SCH-56582 (10 microM) and FPT III (10 miroM), which inhibit p21Ras activation; and Calphostin C (0.2 microM), an inhibitor of protein kinase C. These agents also inhibited Hex A-induced cell proliferation in bovine ASMC. These data suggest that Hex A activates p44/42(MAPK) in a p21Ras- and PKC-dependent manner and that this activation mediates Hex A- induced mitogenesis in bovine ASMC.     

MCP-1-dependent signaling in CCR2(-/-) aortic smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a mediator of inflammation that has been implicated in the pathogenesis of a wide variety of human diseases. CCR2, a heterotrimeric G-coupled receptor, is the only known receptor that functions at physiologic concentrations of MCP-1. Despite the importance of CCR2 in mediating MCP-1 responses, several recent studies have suggested that there may be another functional MCP-1 receptor. Using arterial smooth muscle cells (SMC) from CCR2(-/-) mice, we demonstrate that MCP-1 induces tissue-factor activity at physiologic concentrations. The induction of tissue factor by MCP-1 is blocked by pertussis toxin and 1,2-bis(O-aminophenyl-ethane-ethan)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, suggesting that signal transduction through the alternative receptor is G(alphai)-coupled and dependent on mobilization of intracellular Ca(2+). MCP-1 induces a time- and concentration-dependent phosphorylation of the mitogen-activated protein kinases p42/44. The induction of tissue factor activity by MCP-1 is blocked by PD98059, an inhibitor of p42/44 activation, but not by SB203580, a selective p38 inhibitor. These data establish that SMC possess an alternative MCP-1 receptor that signals at concentrations of MCP-1 that are similar to those that activate CCR2. This alternative receptor may be important in mediating some of the effects of MCP-1 in atherosclerotic arteries and in other inflammatory processes.     

Haemorrhagic shock in mice--intracellular signalling and immunomodulation of peritoneal macrophages' LPS response

Haemorrhagic shock leads to decreased proinflammatory cytokine response which is associated with an increased susceptibility to bacterial infections. In the present study, the effect of GM-CSF on lipopolysaccharide (LPS)-induced TNF-alpha release and MAPkinase activation was analysed on the background of a possible immunostimulating activity of this substance. Male BALB/c mice were bled to a mean arterial blood pressure of 50 mmHg for 45 min followed by resuscitation. Peritoneal macrophages were isolated 20 h after haemorrhage and incubated with 10 ng/ml GM-CSF for 6h before LPS stimulation. TNF-alpha synthesis was studied in the culture supernatants using ELISA. Phosphorylation of ERK, p38MAPK and IkappaBalpha was detected by Western blotting. LPS-induced TNF-alpha production of peritoneal macrophages was significantly decreased 20 h after haemorrhage in comparison to the corresponding cells of sham-operated mice. In parallel the phosphorylation of IkappaBalpha was less in LPS-stimulated peritoneal macrophages from haemorrhagic mice. LPS-induced phosphorylation of ERK1/2 was also decreased in peritoneal macrophages isolated after haemorrhagic shock. In contrast, p38MAPK was phosphorylated more intensely after LPS-stimulation in macrophages collected from shocked mice. GM-CSF incubation elevated LPS-induced TNF-alpha response of macrophages from both sham-operated and shocked mice which was accompanied by an elevated IkappaB and ERK phosphorylation. In general, GM-CSF treatment in vitro enhanced peritoneal macrophages LPS-response both in terms of TNF-alpha synthesis and IkappaB and MAPK signalling, but the levels always stayed lower than those of GM-CSF-treated cells from sham-operated animals. In conclusion, GM-CSF preincubation could partly reactivate the depressed functions of peritoneal macrophages and may therefore exert immunostimulating properties after shock or trauma.     

Bacterial endotoxin stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms

Bacterial endotoxin [lipopolysaccharide (LPS)] stimulates macrophages to sequentially release early [tumor necrosis factor (TNF)] and late [high mobility group box 1 (HMGB1)] proinflammatory cytokines. The requirement of CD14 and mitogen-activated protein kinases [MAPK; e.g., p38 and extracellular signal-regulated kinase (ERK)1/2] for endotoxin-induced TNF production has been demonstrated previously, but little is known about their involvement in endotoxin-mediated HMGB1 release. Here, we demonstrated that genetic disruption of CD14 expression abrogated LPS-induced TNF production but only partially attenuated LPS-induced HMGB1 release in cultures of primary murine peritoneal macrophages. Pharmacological suppression of p38 or ERK1/2 MAPK with specific inhibitors (SB203580, SB202190, U0126, or PD98059) significantly attenuated LPS-induced TNF production but failed to inhibit LPS-induced HMGB1 release. Consistently, an endogenous, immunosuppressive molecule, spermine, failed to inhibit LPS-induced activation of p38 MAPK and yet, still significantly attenuated LPS-mediated HMGB1 release. Direct suppression of TNF activity with neutralizing antibodies or genetic disruption of TNF expression partially attenuated HMGB1 release from macrophages induced by LPS at lower concentrations (e.g., 10 ng/ml). Taken together, these data suggest that LPS stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms.     

Mechanoregulation of monocyte chemoattractant protein-1 expression in rat vascular smooth muscle cells

The authors have previously shown that arterial wall strain mediates the development of vessel wall inflammation in experimental hypertension. The current studies explore the mechanoregulation of monocyte chemoattractant protein-1 (MCP-1), a potent pro-inflammatory chemokine, by mitogen-activated protein kinases (MAPK) and oxidative stress. Rat aortic smooth muscle (RASM) cells were subjected to cyclic strain on a uniform biaxial strain device. Strain rapidly activated both ERK1/2(MAPK) and p38(MAPK), with peak activation at 5 min. Strain induced a twofold increase in MCP-1 mRNA, which was attenuated by PD 98059, a specific ERK1/2(MAPK) inhibitor, and SB 203580, a specific p38(MAPK) inhibitor. Cyclic strain also increased production of superoxide anion via an NADPH oxidase-dependent mechanism. To assess the potential role of reactive oxygen species in MAPK activation, cells were stretched in the presence of N-acetylcysteine, which had no effect on p38(MAPK) activation, but significantly inhibited ERK1/2(MAPK) activation and MCP-1 expression. In conclusion, redox-sensitive activation of ERK1/2(MAPK) and redox-insensitive activation of p38(MAPK) regulate straininduced MCP-1 expression in RASM cells. These findings define a role for MAPK signal transduction in establishing a pro-inflammatory state in the arterial wall, and thus implicate a potential molecular link between arterial wall strain and atherosclerosis.     

Tumor necrosis factor-alpha up-regulates the expression of CCL2 and adhesion molecules of human proximal tubular epithelial cells through MAPK signaling pathways

Both circulating and urinary tumor necrosis factor (TNF)-alpha levels have been shown to increase in inflammatory chronic kidney diseases and TNF-alpha can induce secretion of other inflammatory mediators from many cell types. Chemokine, mononuclear chemoattractant protein-1 (CCL2/MCP-1), and cell surface adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in renal proximal tubular epithelial cells (PTEC) are important for promoting recruitment and adhesion of infiltrating macrophages and lymphocytes to inflamed renal tissue. This study aimed to investigate the effect of TNF-alpha on the expression of these inflammation-related molecules of human PTEC and the underlying intracellular mitogen-activated protein kinase (MAPK) regulatory signaling mechanisms. Cytokine expression profile of TNF-alpha-activated PTEC was assayed by protein array. The concentration of CCL2 was analyzed by ELISA, while the expression of cell surface ICAM-1 and VCAM-1 and intracellular phosphorylated p38 MAPK, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) was assessed using flow cytometry. TNF-alpha could significantly induce CCL2, ICAM-1 and VCAM-1 expression of PTEC. Selective inhibitors of p38 MAPK (SB203580), JNK (SP600125) and ERK (PD98059) could suppress TNF-alpha-induced CCL2 and ICAM-1 expression, while only p38 MAPK and ERK inhibitors could suppress TNF-alpha-induced VCAM-1 expression. JNK inhibitor was found to up-regulate VCAM-1 expression but did not elicit any additive effect with TNF-alpha on VCAM-1 expression. Moreover, p38 MAPK inhibitor was found to abrogate the TNF-alpha-induced ERK phosphorylation, suggesting that there was a one-way interaction between p38 MAPK and ERK pathways during the TNF-alpha activation. TNF-alpha can play a crucial role in the immunopathogenesis of nephritis by the induction of CCL2, ICAM-1 and VCAM-1 expression via the activation of the intracellular MAPK signaling pathway, which may contribute to macrophage and lymphocyte infiltration.     

细胞外信号调节激酶在TPPB促进PC12产生可溶性淀粉样前体蛋白中的作用

目的：观察在蛋白激酶C(PKC)激动剂TPPB促进可溶性淀粉样前体蛋白(sAPPα)释放过程中参与的信号转导通路。方法:以1 μmol/L的TPPB作用于PC12细胞3 h，同时加入信号转导通路的抑制剂，Western印迹法检测上清液内sAPPα的含量和细胞外信号调节激酶(p42/44MAPK)及磷酸化的p42/44MAPK的表达。结果:1 μmol/L的TPPB作用于PC12细胞3 h可以显著增加上清液内sAPPα的含量，细胞外信号调节激酶抑制剂U0126、c-Jun氨基末端激酶抑制剂SP600125和蛋白酪氨酸激酶抑制剂genistein可以部分消除此作用；而p38MAPK抑制剂SB203580对sAPPα的含量无显著影响。1 μmol/L的TPPB可以使磷酸化的p42/44MAPK表达增加，而对总的p42/44MAPK无显著影响。结论:细胞外信号调节激酶、c-Jun氨基末端激酶和蛋白酪氨酸激酶可能参与TPPB促进sAPPα生成的过程。     

Bacterial lipopolysaccharide induces proliferation of IL-6-dependent plasmacytoma cells by MAPK pathway activation

Mouse plasmacytomas are appropriate models to study the biology of human multiple myeloma (MM). Growth of murine interleukin-6 (IL-6)-dependent hybridoma/plasmacytoma lines can be stimulated by bacterial lipopolysaccharides (LPS). However, the molecular mechanisms of this phenomenon are still not elucidated. In this study the in vitro action of bacterial LPS on the mouse IL-6-dependent B9 hybridoma/plasmacytoma cell line and two IL-6-dependent hybridomas was investigated. The involvement of different signal transduction pathways was established using specific kinase inhibitors in proliferation assays and immunoblotting analysis of the kinase activity. Selective mitogen-activated protein kinase (MAPK) kinase inhibitor PD989059 inhibited both IL-6- and LPS-induced B9 cell proliferation. In contrast, in H187 and H188 cells, PD98059 inhibited only LPS-, but not IL-6-stimulated cell growth. The kinetics of MAPK activation in all cell lines showed that phosphorylation of p42 MAPK (encoded as ERK2) but not of p44 MAPK (ERK1), was considerably increased after treatment with LPS. We found that in H187 and H188 hybridomas IL-6 induced proliferation by a different STAT3-dependent mechanism. This study demonstrates the key role of the MAPK pathway in LPS-stimulated growth of mouse IL-6-dependent plasmacytoma cells. These findings suggest the presence of signaling mechanism in MM cells inducible by bacterial mitogens and possibly mediated by Toll-like receptors (TLR) – evolutionarily conserved molecules playing a central role in the microbial recognition and initiation of the cellular innate immune response.